Crosslinked polysaccharides and related methods

ABSTRACT

Methods of forming cross-linked polysaccharides are disclosed in which one or more polysaccharides are dissolved in solution, gelled, modified to have a desired concentration, and subsequently irradiated. The irradiation of the gel crosslinks the polysaccharide(s) present. The disclosed techniques may be applied to various polysaccharides, including but not limited to agarose and/or hyaluronic acid.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 62/783,630, filed Dec. 21, 2018, the contents of which areincorporated by reference herein.

BACKGROUND

Crosslinking is the process of chemically joining two or more polymerchains together through a covalent or ionic bond. Various mechanicalproperties of a polymer can be modified by crosslinking. For example,crosslinking a material to a low crosslink density can decrease theviscosity of polymer melts, while crosslinking to an intermediatecrosslink density can transform a gummy polymer into a material withelastomeric properties and potentially high strength. In some cases,very high crosslink densities can cause a material to become rigid orglassy. Numerous crosslinking techniques are known, including processesthat rely on heat, pressure, change in pH, or radiation to initiate thecrosslinking process.

SUMMARY

This disclosure relates to methods of crosslinking polysaccharides aswell as the resulting crosslinked compositions. In particular, thesubject disclosure describes methods of irradiating polysaccharides,particularly agarose, while in a gelled state to achieve a desired levelof crosslinking.

As used herein, the term “polysaccharide” refers to a polymericcarbohydrate having the general formula C_(x)(H₂O)_(y), such as, forexample, starch, dextrin, cellulose, hemicellulose, polydextrose,inulin, beta-glucan, pectin, psyllium husk mucilage, mannan,beta-mannan, carob, fenugreek, guar, tara gum, glucomannan, gum acacia,karaya, tragacanth, arabinoxylan, gellan, xanthan, alginate, agarose,carrageenan, agar, hyaluronic acid, chitin, and chitosan. Many exampleembodiments in which the polysaccharide agarose is used are described indetail herein. However, the subject disclosure is not intended to be solimited. Specifically, although examples in which agarose is used, anyother suitable type of polysaccharide may alternatively or additionallybe used. For example, embodiments in which hyaluronic acid is used arealso of interest and described in detail herein.

Although irradiation techniques have previously been applied to somepolysaccharides for crosslinking purposes, this type of crosslinkingprocess (as previously performed) has many disadvantages. In particular,at low concentrations in water, most polysaccharides will degrade fromthe effects of irradiation. Degradation can also occur if thepolysaccharide is irradiated in a dry state (i.e., with a waterconcentration of less than 5%). Crosslinking of the polysaccharide thusoccurs with only minimal or no degradation within a particularconcentration range. Below or above such a concentration ofpolysaccharide, degradation can occur and may be quite significant.Degradation of the polysaccharide can take various forms, includingbreakage of one or more glycosidic linkages of the polysaccharide. Asset forth more fully below, techniques are described herein tofacilitate competitive crosslinking of polysaccharides, particularlyagarose, as well as other polysaccharides, such as hyaluronic acid.

Previous attempts at crosslinking polysaccharides by irradiationgenerally involved irradiating the polysaccharide while in a paste-likestate. As used herein, the term “paste-like” refers to a polysaccharidewith a water concentration less than about 90% by weight and/or volume.As opposed to gelled polysaccharides, which have a somewhat orderedstructure, paste-like polysaccharides do not necessarily have an orderedstructure and can exhibit non-uniform mechanical properties throughout.As explained more fully herein, irradiating a polysaccharide in anordered gel state by irradiation may produce a uniquely crosslinkedstructure as opposed to irradiating a paste-like polysaccharide.

It is important to note that, to the knowledge of the subjectinventor(s), agarose has not previously been crosslinked by irradiationtechniques. There are a few reasons that the disclosed techniques havebeen not been attempted. Firstly, the particular concentration range ofagarose needed to facilitate crosslinking as opposed to degradation isdifficult to achieve. Also, there previously had been limited use foragarose gels having an agarose concentration within the useful range forcrosslinking via irradiation. Thus, the disclosed compositions andtechniques are believed to be new and have not previously been easilyachievable. Additionally, the present disclosure describes newbeneficial uses for the described irradiated and crosslinked agarosematerials, which were previously unknown.

Crosslinked polysaccharides have many useful properties. For example,crosslinked agarose is significantly more robust than non-crosslinkedagarose. Accordingly, crosslinked agarose has promising potential foruses in many applications, including as dermal filler, cartilagereplacement, sutures, surgical fabric, wound care materials, tissue andbone scaffolding, and/or drug delivery vehicles. Crosslinking agarose ingel form via irradiation also provides a number of advantages which mayonly be possible using the disclosed techniques. For example, thedisclosed techniques may provide: good control over agaroseconcentration, a wide range of various agarose concentrations, and/orgel that can be formed in a predictable size and shape. As described inmore detail below, gels and other articles formed from the disclosedcrosslinked agarose may be used for any purpose, including cosmetic,reconstructive, and/or therapeutic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be more fully understood with reference tothe accompanying drawings.

FIG. 1 illustrates an exemplary method of preparing a crosslinkedagarose gel, in accordance with various embodiments of the subjectdisclosure.

FIG. 2 illustrates an exemplary method of preparing a crosslinkedhyaluronic acid gel, in accordance with various embodiments of thesubject disclosure.

DETAILED DESCRIPTION

As shown in FIG. 1, method 200 includes dissolving agarose in a solventto form a solution containing agarose (Block 202). As used herein, theterm “agarose” refers to a compound based on the following polymericstructure:

The agarose used in the disclosed methods and compositions may becommercially obtained or prepared by a user. The disclosed agarose may,in some embodiments, include one or more crude, purified, derivatized ormodified agars or agaroses. For example, in certain embodiments, theagarose is selected from agarose, purified agarose, modified agarose,and derivatized agarose. The agarose may also be used as mixtures withother compatible polymers and additives such as agar, carrageenan,chitosan, alginate, gelatin, hyaluronic acid, collagen, in someembodiments. In select embodiments, the agarose is unmodified ormodified agarose, and/or derivatized agarose. In certain embodiments,the agarose is Gracilaria-derived agarose. Gracilaria-derived agarosehas a higher methoxy content than agarose derived from other sources(e.g., Gelidium). Agaroses from other seaweeds, for example, Pterocladiaor Gelidiella may also be used as the disclosed agarose.

Any suitable solvent may be used to dissolve the agarose. For example,in some embodiments, the agarose may be dissolved in water with orwithout non-aqueous liquid(s) present. Example non-aqueous liquids thatmay be used include but are not limited to glycerine and a glycol. Insome embodiments, the agarose may be dissolved in sufficient solvent toproduce a solution with at least 1%, 3%, 5%, 10%, 12%, 15% or moreagarose by weight. In these and other embodiments, a solution havingbetween 1% and 15%, between 3% and 10%, or approximately 5% agarose byweight may be prepared. In some embodiments, the solvent may be heatedto facilitate dissolution of the agarose.

If appropriate for the intended application, one or more additives mayalso be added to the solution containing agarose (Block 203). Ifpresent, additives in liquid and/or solid form may be added to thesolution containing agarose. In some embodiments, (crosslinked ornon-crosslinked) hyaluronic acid may be added to the solution containingagarose. In some such embodiments, the hyaluronic acid may be added as asolution or particles to the solution containing agarose. In these andother embodiments, hydroxyapatite may be added to the solutioncontaining agarose. Hydroxyapatite may be especially useful as anadditive in embodiments in which the resulting crosslinked agarose gelis to be used for dermal filling applications, bone tissue engineering,and the like. Other example additives that may be used include porons,such as particles, beads, threads, rods, or other possible structures.In some such embodiments, the porons may be incorporated into the geland may thereafter be physically removed from the gel or dissolved andleached from the gel after it has formed to create pores and/or passageswithin the gel.

Method 200 continues with forming an agarose gel from the dissolvedagarose (Block 204). The dissolved agarose may be gelled according toany known technique, including chemical crosslinking. In someembodiments, gelling may be accomplished by filling a mold or othercasting device with a solution containing agarose and allowing thesolution to gel. In some embodiments, the agarose may be gelled at aroom temperature, or a temperature higher or lower than roomtemperature. After gelling, the agarose gel may have an agaroseconcentration of at least 0.1%, 1%, 3%, 5%, 7%, 10%, 12%, 15%, or moreby weight.

In some embodiments, forming an agarose gel (Block 204) includes gellingthe solution containing agarose as a foam or as an open matrixstructure. In select embodiments, the solution containing agarose isapplied as a coating on an implant or other substrate and then gelledwhile on the substrate. In these and other embodiments, the solutioncontaining agarose may be imbibed into an absorbent material (e.g., abandage or sponge) and then gelled on and/or in the absorbent material.In select embodiments, the solution containing agarose may be gelled viaan extrusion process, thereby forming a gel having a well-definedstructure (e.g., threads, rods, tubes, or other structures having adesired cross-section). In these and other embodiments, the solutioncontaining agarose may be gelled as beads.

Method 200 continues with optionally adjusting the concentration ofagarose in the agarose gel (Block 206). As previously explained, agarosegels may be crosslinked by irradiation to yield crosslinked agarose gelswith numerous advantageous properties. Without wishing to be bound bytheory, it is believed that crosslinking by irradiation is best suitedfor agarose gels having a particular agarose concentration. For example,agarose gels having an agarose concentration of between 10% and 80% arebelieved to be within a desirable range to crosslink by irradiationtechniques. Method 200 thus includes the optional step of adjusting theagarose concentration in the gel, if desired, to be within a rangewell-suited to crosslinking by irradiation. In some embodiments, theagarose concentration may be adjusted to be between 10% and 80% byweight. In these and other embodiments, the agarose concentration may beadjusted to be between 20% and 60%, between 30% and 50%, or between 35%and 45% by weight. The agarose concentration of the gel may be adjustedusing any suitable technique. For example, in some embodiments, theagarose gel may be fully or partially dehydrated. In some suchembodiments, the partially or fully dehydrated agarose gel may berehydrated (partially or fully) to achieve the desired agaroseconcentration.

Method 200 continues with optionally incorporating one or more additivesinto the agarose gel (Block 208). Example additives and relative weightpercentages can be any additives previously discussed herein. Inparticular embodiments, hyaluronic acid (HA) is incorporated into theagarose gel. Without wishing to be bound by theory, the HA may crosslinkwith itself and/or with the agarose during processing, which may producea crosslinked gel composition having unique properties. Such a gel mayalso be affected by exposure to hyaluronidase. It should be noted thatif one or more additives are incorporated into the agarose gel, the oneor more additives may be added prior to or after adjusting theconcentration of agarose in the agarose gel (pursuant to Block 206), ifthe agarose concentration is adjusted.

Method 200 continues with irradiating the agarose gel to form acrosslinked agarose gel (Block 210). The agarose gel may be irradiatedusing any suitable technique, such as processes that employ gammaradiation, x-ray or beta radiation (e.g., electron beam “e-beam”processing). Numerous types of irradiating devices are known in the artand may be used to irradiate agarose gel according to the disclosedmethods. The agarose gel may be irradiated with any suitable amount ofradiation, depending on the desired specifications of the resultingcrosslinked agarose gel. For example, in some embodiments, the agarosegel may be dosed with at least 5 kilograys (kGy), 10 kGy, 20 kGy, 30kGy, 40 kGy, 50 kGy, 60 kGy, 70 kGy, 80 kGy, 90 kGy, 100 kGy, or more.In select embodiments, the agarose gel is irradiated with between 10 and100 kGy, between 20 and 80 kGy, or between 40 and 60 kGy.

The resulting agarose gel crosslinked via the disclosed irradiationprocess may have various mechanical properties. For example, in someembodiments, the crosslinked agarose gel may exhibit increased strengthas compared to an agarose gel formed according to the same techniquethat has not been crosslinked. Additionally, the crosslinked agarose gelmay, in some embodiments, no longer be thermally reversable. In otherwords, the crosslinked agarose gel may not melt upon exposure to anamount of heat that would cause a similar non-crosslinked agarose gel tomelt. Numerous other mechanical properties of the disclosed crosslinkedagarose gel are possible and contemplated herein.

Method 200 of FIG. 1 includes optionally incorporating one or moreadditives into the crosslinked agarose gel (Block 212). One or moreadditives may be incorporated into the crosslinked agarose gel if, forexample, the additive(s) might not tolerate irradiation. In embodimentsin which the one or more additives incorporated into the crosslinkedagarose gel are in liquid form, the one or more additives may be infusedinto the gel. In embodiments in which the one or more additivesincorporated into the crosslinked agarose gel are solid(s), the one ormore additives may be loaded into the gel matrix. Example liquidadditives include but are not limited to pharmaceutical agents or othertypes of beneficial agents. Example solid additives include but are notlimited to cells, tissue, pharmaceutical and/or beneficial agents.

Method 200 of FIG. 1 concludes with optionally administering thecrosslinked agarose gel to a patient (Block 214). The disclosedcrosslinked agarose gels may be administered in any desired structure.For example, in some embodiments, the crosslinked gels may be used asis, such as in the form of sheets, threads, rods, cast structures,matrices, or other defined structures. In these and other embodiments,the crosslinked agarose gels may be ground or chopped to create smallerpieces or particles. In further embodiments, the crosslinked agarosegels may first be dehydrated and then used as is or chopped or ground upafter dehydration. In yet other embodiments, the disclosed crosslinkedagarose gels may be combined with non-crosslinked gels or othermaterials to create hybrid structures. Numerous configurations andvariations are possible and contemplated herein.

In some embodiments, the crosslinked agarose gel is administered to apatient transdermally via a needle. In some such embodiments, the gelmay be prepared for use by an aseptic fill process (e.g., a process inwhich the agarose gel is loaded into a delivery device in a sterilemanner). In embodiments in which an aseptic fill process is used, theremay be no need for a terminal sterilization step to occur in which theagarose gel is sterilized after at least some packaging has taken place.The delivery technique can be selected based on the intended use of thecrosslinked agarose gel. In select embodiments, the crosslinked agarosegel may be used for dermal fill, reconstruction, and/or scaffoldingapplications. In select embodiments, the disclosed agarose gels are usedfor one or more of the following: filling in wrinkles, fine lines, ordeep creases, improving skin imperfections, such as scars, adding volumeto lips or cheeks, contouring the jaw line, or adjusting the appearanceof any other body part, such as rhinoplasty. Countless other uses forthe disclosed crosslinked agarose gels are possible and contemplatedherein.

In some embodiments, the disclosed agarose gel compositions areadministered to a patient at concentrations of at least 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10% by weight. In these and other embodiments, theagarose gel crosslinked by irradiation may be mixed with one or moreother types of hydrogels. For example, in some embodiments, hyaluronicacid (HA) is mixed with the crosslinked agarose gel prior toadministration to a patient.

The disclosed techniques and compositions may provide numerousadvantages over alternative preparation and sterilization procedures.Notably, crosslinking an agarose by irradiating an agarose gel having aconcentration within a desired range can produce a robust crosslinkedagarose well-suited to application within the human body. Agarose gelsprepared according to the disclosed methods may have improved tactileeffects in the body. For example, the disclosed agarose gels (formedfrom an agarose gel crosslinked by irradiation) may be firmer thanconventional agarose gels. Agarose gels prepared according to thedisclosed methods may, in some embodiments, be sterilizable by heatwithout significant loss of gel structure. Due to the nature of thepresently disclosed agarose gels, agarose gels crosslinked by thedisclosed techniques may have increased overall residence time in thebody, thereby affording additional time before follow-up procedures areneeded to replenish gel that is consumed by the body.

Example Embodiments with Hyaluronic Acid

As previously explained, although numerous examples are disclosed inwhich agarose is used as the polysaccharide crosslinked by irradiation,the subject disclosure also extends to embodiments in which otherpolysaccharides are crosslinked via irradiation techniques while in agelled state. For example, in some embodiments, a hyaluronic acid gel isformed and subsequently irradiated to form a crosslinked hyaluronic acidgel. Method 300 of FIG. 2 describes an example method of crosslinking ahyaluronic acid gel using irradiation techniques.

As shown in FIG. 2, method 300 includes forming a hyaluronic gel (Block302). Forming a hyaluronic acid gel can be accomplished using anysuitable method known to those skilled in the art. Method 300 continueswith optionally adjusting the concentration of hyaluronic acid in thehyaluronic acid gel Block 304). If performed, the concentration ofhyaluronic acid may be adjusting using any technique described hereinwith respect to Block 206 of method 200. Additionally, the concentrationof hyaluronic acid may be adjusted to any concentration previouslydescribed for agarose in method 200 (e.g., 10%-80%, 20%-60%, 30%-50%, or35%-45% by weight). The hyaluronic acid gel may then be irradiated toform a crosslinked hyaluronic acid gel (Block 306). Any technique may beused to irradiate the hyaluronic acid gel, including techniquesdescribed in method 200, Block 210. One or more additives may optionallybe incorporated into the hyaluronic acid gel prior to or afterirradiation, as desired. Any additives previously discussed herein maybe incorporated into the hyaluronic acid gel. The crosslinked hyaluronicacid gel may then be administered to a patient (Block 308). Anyappropriate administration technique may be used to administer thecrosslinked hyaluronic acid gel to a patient, including those previouslydiscussed with respect to Block 214 of method 200.

Example Embodiments with Agarose and Hyaluronic Acid

A particular example embodiment in which a mixture of agarose gel andhyaluronic acid is crosslinked via irradiation is described in detailbelow.

Hyaluronic acid (that has not been crosslinked) tends to form a thick,gel-like solution, even at relatively high concentrations. In contrastto an agarose gel, which adopts the particular shape/structure in whichit was gelled, hyaluronic acid behaves more like a viscous solution andadopts the shape of a vessel in which it is contained. In other words,while agarose gels can typically retain a given shape or structure,hyaluronic acid gels tend to be more malleable. While crosslinkinghyaluronic acid via irradiation may have useful applications, theseapplications may not require the crosslinked hyaluronic acid to have awell-defined structure or shape. However, a mixture of agarose andhyaluronic acid may form a gel having a defined structure and, afteradjusting the water content to a suitable amount (if necessary) themixture of agarose gel and hyaluronic acid may be crosslinked viairradiation (using any of the techniques previously described herein) toform a crosslinked gel having a defined shape/form. Without wishing tobe bound by theory, it is believed that a gel containing agarose andhyaluronic acid that is crosslinked via irradiation may have a gelmatrix that exhibits unique properties.

To produce a crosslinked gel containing agarose and hyaluronic acid, asolution containing agarose and hyaluronic acid may be created. Forpurposes of illustration, a solution containing between 1 and 2% agaroseby weight and approximately 6% hyaluronic acid by weight may beproduced. However, in other embodiments, the concentrations of agaroseand/or hyaluronic acid may be increased or decreased and/or the relativeconcentrations of each may be altered. The solution may then be gelledto form a particular shape or structure. After gel formation, the gelmay be partially dehydrated to have an agarose and hyaluronic acidconcentration within an acceptable range to crosslink using irradiation.The gel may then be irradiated to crosslink the agarose and hyaluronicacid. In this example embodiment, crosslinks may form between agarosechains, between hyaluronic acid chains, and/or between agarose chainsand hyaluronic acid chains. Numerous configurations and variations arepossible. The crosslinked gel may then be partially rehydrated (ifdesired) and administered to a patient with or without additionalfurther processing. Additionally, in some embodiments, the crosslinkedgel may be exposed to a hyaluronidase enzyme to degrade the hyaluronicacid in the crosslinked gel. In some such embodiments, the resulting gelmay retain its form or shape even after the hyaluronic acid has beenpartially or fully degraded. In other embodiments, the resultant gel maylose some or all of its strength, form or shape after the hyaluronicacid has been partially or fully degraded.

The features and advantages described herein are not all-inclusive and,in particular, many additional features and advantages will be apparentto one of ordinary skill in the art in view of the present disclosure.Persons skilled in the relevant art can appreciate that manymodifications and variations are possible in light of the abovedisclosure.

The invention claimed is:
 1. A method of forming a crosslinked agarosegel, the method comprising: dissolving an agarose in a solvent to form asolution containing agarose; gelling the solution containing agarose toform a gelled agarose; modifying the gelled agarose to have an agaroseconcentration of between 10% and 80%; irradiating the gelled agarosehaving an agarose concentration of between 10% and 80% to form acrosslinked agarose gel.
 2. The method of claim 1, wherein the agaroseis selected from the group consisting of: purified agarose, modifiedagarose, and derivatized agarose.
 3. The method of claim 1, wherein thesolvent comprises water.
 4. The method of claim 3, wherein the solventcomprises glycerine or a glycol.
 5. The method of claim 1, whereinmodifying the gelled agarose comprises at least partially dehydratingthe gelled agarose to yield a gelled agarose having an agaroseconcentration of between 10% and 80%.
 6. The method of claim 1, whereinthe gelled agarose has an agarose concentration of between 20% and 60%.7. The method of claim 1, wherein irradiating is accomplished byexposure to one or more of the following: gamma radiation, x-rays, orbeta radiation.
 8. The method of claim 1, wherein the gelled agarose isirradiated with at least 5 kilograys (kGy) of radiation.
 9. The methodof claim 1, further comprising administering the crosslinked agarose gelto a patient.
 10. The method of claim 9, further comprising mixing thecrosslinked agarose gel with hyaluronic acid prior to administering tothe patient.
 11. The method of claim 1, wherein gelling the solutioncontaining agarose to form a gelled agarose comprises gelling thesolution containing agarose as a foam or as an open matrix structure.12. The method of claim 1, wherein gelling includes applying thesolution containing agarose as a coating on a substrate and then gellingthe solution while on the substrate.
 13. The method of claim 1, whereingelling includes imbibing the solution containing agarose into anabsorbent material and then gelling the solution containing agarose onor in the absorbent material.
 14. The method of claim 1, wherein gellingthe solution containing agarose is accomplished through an extrusionprocess.
 15. The method of claim 1, further comprising incorporating oneor more additives into the crosslinked agarose gel.
 16. The method ofclaim 15, wherein the one or more additives are selected from the groupconsisting of pharmaceutical or beneficial agents, cells, and tissue.17. The method of claim 1, further comprising incorporating one or moreadditives into the solution containing agarose.
 18. The method of claim17, wherein the one or more additives comprise hyaluronic acid.